How to make Coenzyme Q10

Abstract

The present invention provides a method for producing coenzyme Q10 that can be operated economically and stably by effectively concentrating the culture suspension of coenzyme Q10-producing microorganisms and minimizing the loss of coenzyme Q10 before extraction and crushing. The present invention is a method for producing coenzyme Q10 having a filtration step of heating the culture suspension of coenzyme Q10-producing microorganisms to above 35°C and passing the liquid through a porous membrane. In order to increase the average permeability, it is preferred to implement at least one regeneration treatment in which the filter device is sealed and pressurized during the liquid passing treatment of the culture suspension and then the pressure is released; or implement at least one regeneration treatment in which the filtrate outlet side is flowed into the medium during the liquid passing treatment of the culture suspension for a certain period of time and then the normal filtration process is resumed.

Description

Technical Field

[0001] The present invention relates to a method for producing coenzyme Q10. More specifically, the present invention relates to a method for producing coenzyme Q10 comprising a filtration step of passing a culture suspension of a coenzyme Q10-producing microorganism through a porous membrane. Background Art

[0002] Coenzyme Q is an essential component widely distributed in organisms from bacteria to mammals, and is widely known as a component of the electron transport chain of mitochondria in cells in organisms. It is known that coenzyme Q functions as a transfer component in the electron transport chain by repeatedly oxidizing and reducing in mitochondria, and reduced coenzyme Q also has an antioxidant effect. For human coenzyme Q, coenzyme Q10 with 10 repeating structures in the side chain of coenzyme Q is the main component, and in organisms, usually about 40 to 90% exists in the form of a reduced form. As the physiological effects of coenzyme Q, the activation of energy production brought about by mitochondrial activation, the activation of cardiac function, the stabilization effect of cell membranes, the protective effect of cells brought about by antioxidant effects, etc. can be listed.

[0003] Most of the coenzyme Q10 currently manufactured and sold is an oxidized form. In recent years, reduced coenzyme Q10, which shows higher oral absorption than oxidized coenzyme Q10, has also been marketed and is gradually being widely used.

[0004] Some methods for producing coenzyme Q10 are known. For example, Patent Document 1 describes a method for producing reduced coenzyme Q10, which comprises culturing a microorganism that produces reduced coenzyme Q10, disrupting the microorganism cells as needed, and then extracting with an organic solvent.

[0005] In addition, Patent Document 2 describes a method for producing Coenzyme Q10, which includes contacting an extract of a Coenzyme Q10-producing microorganism with an adsorbent having aluminum silicate as a main component alone or with a plurality of adsorbents obtained by combining the adsorbent with a different adsorbent. According to the method of Patent Document 2, a method for producing Coenzyme Q10 is described, which can efficiently remove impurities derived from microorganisms from an extract of Coenzyme Q10-producing microorganisms and operate the production process of Coenzyme Q10 in a simple and stable manner.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2008-253271

[0009] Patent Document 2: WO2018 / 003974 Summary of the invention

[0010] Problems to be solved by the invention

[0011] In the above-mentioned conventional methods, there is still room for improvement in order to stably, efficiently and conveniently mass-produce coenzyme Q10.

[0012] For example, in the method of Patent Document 1, since the concentration of the culture suspension of the coenzyme Q10-producing microorganism is low and the water content is high, not only does the equipment in the extraction process need to be large-scale, but it also takes a lot of time to disrupt the microbial cells, which has problems such as poor economic efficiency.

[0013] Patent Document 2 states that the microbial cell culture solution can be appropriately concentrated before extraction, and in the examples, the concentration was performed by centrifugal separation. However, when the concentration is performed using a centrifuge, a portion of the coenzyme Q10-producing microbial cells flows out to the supernatant, which sometimes results in a decrease in yield.

[0014] The present invention is completed in order to solve the above-mentioned problems, and its purpose is to provide a method for producing coenzyme Q10 having a filtration step performed before the extraction step, wherein the filtration step can minimize the loss of coenzyme Q10 in the culture suspension of a coenzyme Q10-producing microorganism, and can concentrate the culture suspension as effectively as possible, and can be operated simply and stably.

[0015] Solution to the problem

[0016] The present inventors have conducted intensive research to solve the above-mentioned problems. As a result, they have found that it is effective to provide a filtration step of passing the culture suspension of the coenzyme Q10-producing microorganism through a porous membrane under a specific temperature condition of 35°C or higher before the extraction step. By adopting the filtration step, the solid content concentration of the culture suspension can be increased, and then the components in the microorganism can be extracted and purified, thereby minimizing the loss of coenzyme Q10 and purifying coenzyme Q10 with good efficiency, thereby completing the present invention.

[0017] That is, the configuration of the method for producing coenzyme Q10 of the present invention is as follows.

[0018] 1. A method for producing coenzyme Q10, comprising heating a culture suspension of a coenzyme Q10-producing microorganism to a temperature of 35° C. or higher and filtering the suspension through a porous membrane.

[0019] 2. The production method according to item 1 above, wherein the heating temperature is 48° C. or higher.

[0020] 3. The production method according to 1 or 2 above, wherein the pH of the culture suspension is in the range of 3 to 7.

[0021] 4. The production method according to any one of 1 to 3 above, wherein the linear velocity of the liquid passing in the filtration step is 0.1 m / s or more.

[0022] 5. The production method according to any one of 1 to 4 above, wherein during the liquid passage treatment of the culture suspension, a regeneration treatment of sealing and pressurizing the filtration device and then releasing the pressure is performed at least once.

[0023] 6. The production method according to 5 above, wherein the pressure during the pressurization is 0.1 to 1 MPa.

[0024] 7. The production method according to 5 or 6 above, wherein the medium used for the pressurization is any one or more of air, nitrogen, water, the culture suspension, and a filtrate obtained by passing the culture suspension through a porous membrane.

[0025] 8. The production method according to any one of 1 to 7 above, wherein after the filtration step is completed, the following operations are repeatedly performed: a washing step of washing the microbial suspension components attached to the porous membrane, and then the filtration step is performed again.

[0026] 9. The production method according to 8 above, wherein the cleaning liquid used for the cleaning is any one or more of water, an alkaline aqueous solution, and an acidic aqueous solution.

[0027] 10. The production method according to 8 or 9 above, wherein the temperature of the cleaning liquid used for the cleaning is 10°C to 90°C.

[0028] 11. The production method according to any one of 1 to 10 above, wherein the porous membrane is any one of a synthetic resin, ceramic, or metal membrane.

[0029] 12. The production method according to 11 above, wherein the porous membrane is a porous membrane made of metal.

[0030] 13. The production method according to 12 above, wherein the metal porous membrane is a cylinder including a titanium oxide-based separation layer and a stainless steel support, and the inner diameter thereof is 5 mm or more.

[0031] 14. The production method according to 13 above, wherein the average pore diameter of the separation layer is 0.01 to 3 μm.

[0032] 15. The production method according to any one of 1 to 14 above, wherein in the liquid passing treatment of the culture suspension, after the liquid is passed through the medium from the filtrate outlet side, the filtration step is resumed.

[0033] 16. The production method according to 15 above, wherein the medium to be introduced is any one or more of air, nitrogen, water, and a filtrate obtained by passing the culture suspension through a porous membrane.

[0034] 17. The production method according to 15 or 16 above, wherein the temperature of the inflowing medium is 10°C to 90°C.

[0035] Effects of the Invention

[0036] According to the present invention, a method for producing coenzyme Q10 can be provided, which can minimize the loss of coenzyme Q10 in the culture suspension of coenzyme Q10-producing microorganisms and effectively concentrate the culture suspension by performing a filtration step of passing the culture suspension of coenzyme Q10-producing microorganisms through a porous membrane before the extraction step. As a result, coenzyme Q10 can be effectively obtained from the perspective of operability and economy, such as reducing the amount of solvent used when extracting or purifying coenzyme Q10 and stabilizing the extraction. DETAILED DESCRIPTION

[0037] Hereinafter, one embodiment of the method for producing coenzyme Q10 of the present invention will be described, but the present invention is not limited thereto.

[0038] The production method of the present invention is characterized in that it has a filtration step of passing a culture suspension of a coenzyme Q10-producing microorganism through a porous membrane while being heated to 35° C. or higher. In the production method of the present invention, coenzyme Q10 is extracted from a microbial cell suspension containing a coenzyme Q10-producing microorganism, or a microbial cell disrupted product or a concentrated solution obtained by concentrating an aqueous suspension of microbial cell disrupted products, using an organic solvent, and further, the purified or purified coenzyme Q10 can be separated / recovered by contacting the solution with an alkaline aqueous solution or water as needed.

[0039] Hereinafter, the production method of the present invention will be described in detail.

[0040] (1) Coenzyme Q10-producing microorganisms used in the present invention

[0041] Coenzyme Q10 exists in an oxidized form and a reduced form. In the present invention, as coenzyme Q10, any one of oxidized coenzyme Q10 and reduced coenzyme Q10 is used as the object, and coenzyme Q10 as a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10 is also its object. In the case where the coenzyme Q10 used in the present invention is a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10, the reduced coenzyme Q10 content ratio is not particularly limited. It should be noted that in this specification, when it is only recorded as coenzyme Q10, regardless of its type, it represents all of oxidized coenzyme Q10, reduced coenzyme Q10, and a mixture of reduced coenzyme Q10 and oxidized coenzyme Q10.

[0042] As the coenzyme Q10-producing microorganism used in the present invention, any of bacteria, yeasts, and molds can be used without limitation as long as it is a microorganism that produces coenzyme Q10 in the microorganism. Specific examples of the above-mentioned microorganisms include Agrobacterium, Aspergillus, Acetobacter, Aminobacter, Agromonas, Acidiphilium, Bulleromyces, Bullera, Brevundimonas, Cryptococcus, Chionosphaera, and Candida. Candida, Cerinosterus, Exisophiala, Exobasidium, Fellomyces, Filobasidiella, Filobasidium, Geotrichum, Graphiola, Gluconobacter, Kockovaella, Kurtzmanomyces, Lalaria, Leucosporidium ucosporidium, Legionella, Methylobacterium, Mycoplana, Oosporidium, Pseudomonas, Psedozyma, Paracoccus, Petromyc, Rhodotorula, Rhodosporidium, Rhizomonas, Rhodobium, Rhodozobium hodoplanes, Rhodopseudomonas, Rhodobacter, Sporobolomyces, Sporidiobolus, Saitoella, Schizosaccharomyces, Sphingomonas, Sporotrichum, Sympodiomycopsis, Sterigmatosporidium, Tapharina,Tremella, Trichosporon, Tilletiaria, Tilletia, Tolyposporium, Tilletiopsis, Ustilago, Udeniomyce, Xanthophllomyces, Xanthobacter, Paecilomyces, Acremonium, Hyhomonus, Rhizobium, Phaffia, Haematococcus and other microorganisms.

[0043] Among them, from the viewpoint of the ease of cultivation and productivity, preferably bacteria or yeast. In bacteria, non-photosynthetic bacteria are more preferably used, and Agrobacterium, Gluconobacter, etc. can be cited as particularly preferred examples, and Schizosaccharomyces, Saitoella, Phaffia, etc. can be cited as particularly preferred examples in yeast.

[0044] It should be noted that, as coenzyme Q10, in order to produce reduced coenzyme Q10, it is preferred to use a microorganism that has a high content of reduced coenzyme Q10 in the produced coenzyme Q10. For example, it is more preferred to use a microorganism in which the content ratio of reduced coenzyme Q10 in coenzyme Q10 after culture (based on weight %) is preferably 70% or more, more preferably 80% or more.

[0045] As the coenzyme Q10-producing microorganism used in the present invention, not only wild strains of the above-mentioned microorganisms can be used, but also mutants and recombinants in which the transcription and translation activities of genes related to the biosynthesis of coenzyme Q10 as the target microorganism or the enzyme activity of the expressed protein are changed or improved.

[0046] By culturing the above-mentioned microorganisms, microbial cells containing coenzyme Q10 can be obtained. The culturing method is not particularly limited, and a culturing method suitable for the target microorganism or suitable for the production of the target coenzyme Q10 can be appropriately selected. The culturing period is not particularly limited, as long as it is a period during which the desired amount of the target coenzyme Q10 is accumulated in the microbial cells.

[0047] In the present invention, it is preferred that the culture suspension of the coenzyme Q10-producing microorganism obtained by the above method is passed through a porous membrane, but it is not limited thereto, and the culture suspension of the coenzyme Q10-producing microorganism obtained by other methods may also be passed through. For example, a liquid (suspension of dried microbial cell fragments) obtained by temporarily performing solid-liquid separation and drying with a filter press and resuspending the microbial cells in water can be used. In addition, as described later, a microbial cell fragment or an aqueous suspension of a microbial cell fragment may also be used. The concentration of microorganisms in the culture suspension passed through the porous membrane is not particularly limited, and is preferably in the range of 0.01 to 10% by weight when converted to the dry weight of the microorganism.

[0048] (2) Filtration process

[0049] In the manufacturing method of the present invention, it is necessary to heat the above-mentioned culture suspension to above 35°C and then pass the liquid through the porous membrane. When it is lower than 35°C, miscellaneous bacteria proliferate in the culture suspension, and there is a hidden danger of reduced filtering effect. The above-mentioned heating temperature can be above 35°C, without any particular limitation. In order to further improve the filtering effect, it is preferably above 40°C, and more preferably above 48°C. In addition, its upper limit is not limited. From the viewpoint of operability and quality, it is preferably below 90°C, and more preferably below 60°C. In the filtration process, the above-mentioned heating temperature does not need to be constant, as long as it is within the above-mentioned range, and the temperature can be changed during the filtration operation.

[0050] In addition, when the porous membrane is passed through, the pH of the culture suspension is preferably in the range of 3 to 7, more preferably in the range of pH 3 to 5, and further preferably in the range of pH 3.5 to 4.5. When the pH of the obtained culture suspension satisfies these ranges, it can be used directly, and in other cases, it can be adjusted to the desired pH using an acid or a base. By making the pH in the above range, not only can the precipitation of inorganic salts in the culture suspension be suppressed, and clogging in the porous membrane be prevented, but also the viscosity of the culture suspension can be reduced, and the filtration process using the porous membrane can be accelerated.

[0051] In the above filtration step, the linear velocity when passing the microorganism culture suspension through the porous membrane is not particularly limited, but is preferably 0.1 m / s or more, more preferably 1 m / s or more, and further preferably 3 m / s or more. The faster the linear velocity, the faster the filtration step can be.

[0052] In the above-mentioned filtration step, the permeation flux of the microorganism culture suspension when passing through the porous membrane is not particularly limited as long as it is not completely blocked. Considering economic aspects such as equipment cost and production volume, the higher the value, the better. The average permeation flux through the filtration step is preferably 0.50 kg / min / m 2 More preferably 1.0 kg / min / m 2 above.

[0053] The type of the porous membrane through which the culture suspension of the coenzyme Q10-producing microorganism is passed is not particularly limited, and preferred examples include porous membranes made of synthetic resins, ceramics, metals, and the like.

[0054] The synthetic resin is not particularly limited as long as it has a molecular weight that can withstand the flow of liquid, and examples thereof include polyethylene, polypropylene, polymethyl methacrylate, polystyrene, and fluorine-containing resins, etc. In consideration of price and availability, polyethylene, polypropylene, and polystyrene are preferred.

[0055] Examples of the ceramics include oxides such as alumina, zirconia, and barium titanate, hydroxides such as hydroxyapatite, carbides such as silicon carbide, nitrides such as silicon nitride, halides such as fluorite, and phosphates. In view of versatility and availability, oxide ceramics are preferred, and alumina is more preferred.

[0056] Examples of the metal include iron, copper, zinc, tin, mercury, lead, aluminum, titanium, titanium oxide, stainless steel, etc. In view of acid resistance, alkali resistance, and strength, stainless steel or titanium oxide is preferred.

[0057] In the manufacturing method of the present invention, from the viewpoint of obtaining a high regeneration effect during the regeneration process, it is preferred to use a metal porous membrane, and specifically, the porous membrane is more preferably equivalent to a separation layer of the filter part composed of titanium oxide, and the outer package (support body) supporting it is composed of stainless steel. In the present invention, the "separation layer (filter part)" is not particularly limited as long as it is a separation layer that does not allow the microbial cells or their broken products in the culture suspension to pass through but allows the water-soluble medium part to pass through, and can be any form of mesh, non-woven fabric, or microporous.

[0058] In the manufacture method of the present invention, the shape of the porous membrane for filtering the culture suspension of coenzyme Q10-producing microorganisms is not particularly limited, and is preferably cylindrical from the perspective of operation and equipment settings. In particular, when the porous membrane is composed of a separation layer (filter portion) and an outer package (support body) supporting it as described above, the hollow body with a separation layer based on titanium oxide is more preferably a tube contained in a cylindrical support body such as stainless steel. Specifically, for example, by making a porous membrane with a hollow microporous body (titanium oxide) inside the outer package (stainless steel), the slurry after concentration can be passed through the hollow portion, and the filtrate is discharged to the outside for filtering / concentration. For the tube used in the present invention, considering the ease of operation, it is preferably lightweight and thin, and on the other hand, considering the solid content concentration and viscosity in the culture suspension, its inner diameter is preferably more than 2mm, more preferably more than 5mm. From the above viewpoint, its upper limit is not particularly limited, considering the weight of the equipment, ensuring the viewpoints such as specific surface area, preferably about 30mm.

[0059] In addition, considering the particle size of the microorganisms in the culture suspension and other solid components from the microorganism culture, the average pore diameter of the above-mentioned separation layer is preferably in the range of 0.01 to 3 μm. In addition, considering productivity, strength, difficulty of clogging, and ease of regeneration, it is more preferably 0.05 μm or more, and preferably 1 μm or less, and more preferably 0.8 μm or less. By making the average pore diameter of the separation layer of the porous membrane in the above range, a high yield can be obtained without leaking the solid components containing coenzyme Q10 into the filtrate.

[0060] In the production method of the present invention, the microbial cell suspension can be concentrated by performing the above-mentioned filtration step. The microorganism concentration in the concentrated microbial cell suspension after the filtration step is not particularly limited, and the concentration step is preferably performed at 0.1 to 25% by weight, converted to the dry weight of the microorganisms. Further, considering the viewpoints of stability and economy, the concentration step is more preferably performed in a range of 10 to 20% by weight.

[0061] In the above-mentioned filtration step, in order to increase the average permeation flux, the temperature may be changed during operation and a regeneration operation may be appropriately introduced.

[0062] When continuing the liquid flow of microorganism culture suspension to porous-film, the solid component coming from the microorganism culture suspension is attached to the inside and surface of the porous-film, and there is a tendency that its filtration rate and permeation flux are progressively reduced. Therefore, in the manufacture method of the present invention, in the liquid flow of the culture suspension to the porous-film, it is preferably implemented at least once that the filter is airtight and pressurized, and then the regeneration process of releasing the pressure. By carrying out this regeneration process, the solid component attached to the inside and surface of the porous-film is discharged outside the filter, and the filtration rate can be restored, and the filtering process is accelerated.

[0063] The above-mentioned regeneration process is a process temporarily (during at least a part of the liquid passing process) performed during the liquid passing of the culture suspension to the porous membrane, and does not mean that the regeneration treatment is performed during the entire process of the liquid passing of the culture suspension. Specifically, for example, during the liquid passing of the culture suspension to the porous membrane, the outlet portion is temporarily closed and the liquid passing of the culture suspension from the inlet portion is continued to be pressurized, and then the outlet portion is opened to restore the pressure in the system. By implementing such an operation, a regeneration process of pressurization and pressure release can be implemented. Alternatively, during the liquid passing of the culture suspension to the porous membrane, either the inlet portion or the outlet portion is temporarily closed, and the medium is injected into the system from the outlet portion or the inlet portion on the opposite side to pressurize, and then the closed inlet or outlet portion is opened to restore the pressure in the system. By implementing such an operation, a regeneration process of pressurization and pressure release can also be implemented. The implementation period of such a regeneration process is, for example, less than 10% of the total period in the liquid passing process, preferably less than 5%, and more preferably less than 2%.

[0064] The method for pressurization is not particularly limited. As the medium used for pressurization, it is preferred to use any one or more of air, nitrogen, water, microbial cell suspension, and filtrate obtained by passing the microbial cell suspension through a filtration membrane (hereinafter, sometimes referred to as filtrate) for pressurization. A more preferred medium is air.

[0065] The pressure when the filtration device is sealed and pressurized is not particularly limited, but is preferably in the range of 0.1 to 1 MPa. In consideration of the cleaning effect, regeneration effect, and safety of the device, it is more preferably 0.2 MPa or more, and more preferably 0.6 MPa or less.

[0066] The time from pressurization to pressure release is not particularly limited, but from the viewpoint of productivity, the regeneration treatment is preferably performed in the shortest possible time, preferably within 5 minutes, more preferably within 1 minute, and further preferably within 20 seconds.

[0067] The above regeneration treatment can be performed at least once during the passage of the culture suspension. However, when the regeneration treatment is added, there are problems such as increased processing time and complicated operation, so the upper limit is, for example, 100 times, preferably 60 times.

[0068] In addition, in the manufacture method of the present invention, after the filtration process is finished, it is preferred to repeat the operation of performing the filtration process again after the cleaning process of cleaning the microbial suspension component attached to the porous membrane, thereby, the porous membrane can be utilized for a long time. As the cleaning solution for cleaning the microbial suspension component attached to the porous membrane, it is not particularly limited, and any one or more of water, alkaline aqueous solution, and acidic aqueous solution can be used. Specifically, the type of cleaning solution can be appropriately selected according to the type of the cleaning object, for example, it is recommended to use an alkaline aqueous solution in the cleaning of organic matter and an acidic aqueous solution in the cleaning of inorganic matter. Preferably, an alkaline aqueous solution and an acidic aqueous solution are used, and more preferably, both cleaning based on an alkaline aqueous solution and cleaning based on an acidic aqueous solution are performed.

[0069] The type of the alkaline aqueous solution is not particularly limited, and examples thereof include ammonia water, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, magnesium oxide, calcium hydroxide, sodium acetate, etc. In view of economic efficiency, sodium hydroxide and potassium hydroxide are preferred. The concentration of the alkaline aqueous solution is also not particularly limited, and in view of operability, it is preferably 5% by weight or less.

[0070] The type of the acidic aqueous solution is not particularly limited, and examples thereof include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, carbonic acid, oxalic acid, aminosulfonic acid, citric acid, hydrogen sulfide, etc. In view of economic efficiency and operability, sulfuric acid, aminosulfonic acid, and citric acid are preferred. The concentration of the acidic aqueous solution is also not particularly limited, and in view of economic efficiency and operability, it is preferably 5% by weight or less.

[0071] The temperature of the cleaning solution during cleaning is preferably high, preferably 10°C or higher, more preferably 40°C or higher, and further preferably 65°C or higher. Cleaning at a higher temperature can improve the regeneration efficiency of the porous membrane. The upper limit is not particularly limited, but is preferably 90°C or lower.

[0072] In the manufacturing method of the present invention, as a method for regenerating the porous membrane, in addition to the regeneration method described above, it can also be a method in which a medium is flowed into the filtrate outlet side during the liquid flow treatment of the culture suspension and the filtration process is restored after the liquid flow. The medium flowing in at this time is not particularly limited, and air, nitrogen, water, filtrate, alkaline aqueous solution, acidic aqueous solution, etc. can be used. From the perspective of economy, safety, and quality, air, nitrogen, water, and filtrate are preferably used.

[0073] The above-mentioned regeneration process is a process temporarily performed (during at least a part of the process) during the perfusion treatment of the culture suspension, and does not mean that the regeneration process is performed during the entire process of the perfusion treatment of the culture suspension. Specifically, for example, in the perfusion treatment of the culture suspension, after the filtrate flows into the medium from the outlet side and reversely perfusion is performed for a certain period of time, the perfusion of the culture suspension from the inlet side is resumed. The timing of the above-mentioned regeneration process varies depending on the allowed processing time, etc., and for example, if the average filtration rate is reduced (about 0.2L / min / m 2 Regardless of the average filtration rate, for example, regeneration is performed every 1 hour.

[0074] In addition, in the above-mentioned regeneration process, the time for flowing into the medium for liquid circulation is not particularly limited, as long as the reduced filtration speed is increased to a given value. For example, it is recommended that one regeneration treatment is usually carried out for more than 5 seconds, preferably for more than 15 seconds, and more preferably for more than 30 seconds.

[0075] The temperature of the medium flowing in to regenerate the porous membrane is not particularly limited, but is preferably 10 to 90° C. In order to avoid a large temperature change in the filtration process and ensure washing recovery, it is more preferably 30° C. or higher, and more preferably 70° C. or lower.

[0076] (3) Crushing as needed

[0077] In the manufacturing method of the present invention, after the above-mentioned filtration step, coenzyme Q10 is extracted using an organic solvent. When extracting coenzyme Q10, coenzyme Q10 can also be directly extracted from the microbial cell culture concentrate obtained by the filtration step of passing the liquid through the porous membrane as described above, and the microbial cells in the concentrate are crushed to prepare a microbial cell crushed product or an aqueous suspension of the microbial cell crushed product, and coenzyme Q10 is preferably extracted from the crushed product or the aqueous suspension of the microbial cell crushed product. Alternatively, the microbial cells can be dried and coenzyme Q10 can be extracted from the dried microbial cells.

[0078] It should be noted that in the present invention, the microbial cell culture solution can be firstly disrupted, and then the culture suspension or aqueous suspension of the obtained microbial cell disrupted product can be passed through a porous membrane to be concentrated. That is, the order of the filtration step (2) and the disruption step (3) is irrelevant. However, it is preferred to perform the filtration step (2) first.

[0079] It should be noted that in the "disruption" of the present invention, surface structures such as cell walls may be lost to the extent that the target coenzyme Q10 can be extracted.

[0080] Examples of the above-mentioned crushing method include physical treatment and chemical treatment.

[0081] Examples of the physical treatment include use of a high-pressure homogenizer, a rotary blade homogenizer, an ultrasonic homogenizer, a French press, a ball mill, or a combination thereof.

[0082] Examples of the chemical treatment include treatment with an acid (preferably a strong acid) such as hydrochloric acid or sulfuric acid, treatment with an alkali (preferably a strong alkali) such as sodium hydroxide or potassium hydroxide, and combinations thereof.

[0083] In the present invention, among the above-mentioned cell disruption methods, physical treatment is more preferred as a pretreatment method for extraction / recovery of coenzyme Q10 from the viewpoint of disruption efficiency.

[0084] In the production method of the present invention, as described above, the culture suspension containing the coenzyme Q10-producing microorganism obtained as described above may be concentrated by the above-mentioned filtration step, and then dried to extract coenzyme Q10 from the dried microbial cells. Examples of dryers for drying the microbial cells in this case include fluidized bed dryers, spray dryers, box dryers, cone dryers, cylindrical vibration dryers, cylindrical stirring dryers, inverted cone dryers, filter dryers, freeze dryers, microwave dryers, etc., or combinations thereof.

[0085] The water concentration in the dried microbial cells is preferably in the range of 0 to 50% by weight. In addition, the dried microbial cells may be further crushed by the crushing method described above, or the crushed microbial cells may be dried to obtain a dried crushed microbial cell.

[0086] (4) Extraction process

[0087] In the production method of the present invention, the organic solvent used for extracting coenzyme Q10 is not particularly limited, and examples thereof include hydrocarbons, fatty acid esters, ethers, alcohols, fatty acids, ketones, nitrogen compounds (including nitriles and amides), and sulfur compounds.

[0088] The hydrocarbon is not particularly limited, and examples thereof include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, etc. Among them, aliphatic hydrocarbons and aromatic hydrocarbons are preferred, and aliphatic hydrocarbons are more preferred.

[0089] The aliphatic hydrocarbon is not particularly limited whether it is cyclic, acyclic, saturated or unsaturated, but saturated aliphatic hydrocarbons are usually preferably used. Generally, aliphatic hydrocarbons having 3 to 20 carbon atoms, preferably 5 to 12 carbon atoms, and more preferably 5 to 8 carbon atoms can be used. Specific examples include propane, butane, isobutane, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (e.g., 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3-trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, 2-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-cyclopentane, methylcyclohexane, ethylcyclohexane, methyl ... Preferably, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane, octane, 2,2,3-trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, More preferably, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane, octane, 2,2,3-trimethylpentane, isooctane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, etc., and further preferably, pentane, hexane, cyclohexane, methylcyclohexane, etc. From the viewpoint of particularly high anti-oxidation effect and versatility, pentane, hexane, and methylcyclohexane are particularly preferred, and pentane and hexane are most preferred.

[0090] As the above aromatic hydrocarbon, there is no particular limitation, and generally, aromatic hydrocarbons having 6 to 20 carbon atoms, preferably 6 to 12 carbon atoms, and more preferably 7 to 10 carbon atoms can be used. As specific examples, for example, benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, isopropylbenzene, mesitylene, tetralin, butylbenzene, p-methylisopropylbenzene, cyclohexylbenzene, diethylbenzene, pentylbenzene, dipentylbenzene, dodecylbenzene, styrene, etc. are listed. Toluene, xylene, o-xylene, m-xylene, p-xylene, ethylbenzene, isopropylbenzene, mesitylene, tetralin, butylbenzene, p-methylisopropylbenzene, cyclohexylbenzene, diethylbenzene, pentylbenzene, etc. are preferably used. Toluene, xylene, o-xylene, m-xylene, p-xylene, isopropylbenzene, and tetralin are more preferably used. Isopropylbenzene is most preferably used.

[0091] The halogenated hydrocarbons are not particularly limited, whether they are cyclic, acyclic, saturated or unsaturated, but acyclic halogenated hydrocarbons are usually preferred, chlorinated hydrocarbons and fluorinated hydrocarbons are more preferred, and chlorinated hydrocarbons are still more preferred.

[0092] In addition, halogenated hydrocarbons having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 2 carbon atoms can be preferably used. Specific examples thereof include dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, hexachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, tetrachloroethylene, 1,2-dichloropropane, 1,2,3-trichloropropane, chlorobenzene, and 1,1,1,2-tetrafluoroethane. Preferred are dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane, etc. More preferred are dichloromethane, chloroform, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, and 1,1,1,2-tetrafluoroethane.

[0093] The fatty acid ester is not particularly limited, and examples thereof include propionate, acetate, formates, etc. Acetate and formates are preferred, and acetate is more preferred. The ester group is not particularly limited, and generally, an alkyl ester having 1 to 8 carbon atoms or an aralkyl ester having 7 to 12 carbon atoms can be used, and an alkyl ester having 1 to 6 carbon atoms is preferred, and an alkyl ester having 1 to 4 carbon atoms is more preferred.

[0094] Specific examples of the propionate ester include methyl propionate, ethyl propionate, butyl propionate, and isoamyl propionate, preferably ethyl propionate.

[0095] Specific examples of the acetate include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, amyl acetate, isoamyl acetate, sec-hexyl acetate, cyclohexyl acetate, benzyl acetate, etc. Preferred examples include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, amyl acetate, isoamyl acetate, sec-hexyl acetate, cyclohexyl acetate, etc. Preferred examples include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, and isobutyl acetate, and most preferred is ethyl acetate.

[0096] Specific examples of the formate ester include methyl formate, ethyl formate, propyl formate, isopropyl formate, butyl formate, isobutyl formate, sec-butyl formate, pentyl formate, etc. Preferred are methyl formate, ethyl formate, propyl formate, butyl formate, isobutyl formate, and pentyl formate. Most preferred is ethyl formate.

[0097] The ethers are not particularly limited, whether they are cyclic, acyclic, saturated or unsaturated, but saturated ethers are usually preferred. Usually, ethers having 3 to 20 carbon atoms, preferably 4 to 12 carbon atoms, and more preferably 4 to 8 carbon atoms can be used. Specific examples of the ethers include diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, ethyl vinyl ether, butyl vinyl ether, anisole, phenethyl ether, butylphenyl ether, methoxytoluene, dibutyl ... alkane, furan, 2-methylfuran, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, etc. Preferred are diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, anisole, phenethyl ether, butylphenyl ether, methoxytoluene, diiso ...isopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl ether, diisopropyl More preferably, diethyl ether, methyl tert-butyl ether, anisole, dimethicone, dimethoxyethane, dimethoxybenzene ... Preferably, the ether is methyl tert-butyl ether, methyl tert-butyl ether, and the most preferably, the ether is methyl tert-butyl ether.

[0098] The alcohol is not particularly limited whether it is cyclic, acyclic, saturated or unsaturated, but saturated alcohols are usually preferred. Alcohols having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms are usually used. Among them, monohydric alcohols having 1 to 5 carbon atoms, dihydric alcohols having 2 to 5 carbon atoms, and trihydric alcohols having 3 carbon atoms are preferred.

[0099] Specific examples of these alcohols include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentanol, tert-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, Monohydric alcohols such as 2-ethyl-1-hexanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, allyl alcohol, propargyl alcohol, benzyl alcohol, cyclohexanol, 1-methylcyclohexanol, 2-methylcyclohexanol, 3-methylcyclohexanol, and 4-methylcyclohexanol; dihydric alcohols such as 1,2-ethanediol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, and 1,5-pentanediol; trihydric alcohols such as glycerol.

[0100] Preferred examples of the monohydric alcohol include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentanol, tert-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-octanol, 2-octanol, 2-ethyl-1-hexanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, benzyl alcohol, cyclohexanol, 1-methylcyclohexanol, 2-methylcyclohexanol, 3-methylcyclohexanol, and 4-methylcyclohexanol. Preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentanol, tert-pentanol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-1-butanol, and cyclohexanol. More preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentanol, tert-pentanol, 3-methyl-2-butanol, and neopentyl alcohol. More preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, 2-methyl-1-butanol, and isopentanol, and most preferred is 2-propanol.

[0101] As the diol, 1,2-ethylene glycol, 1,2-propylene glycol, and 1,3-propylene glycol are preferred, and 1,2-ethylene glycol is most preferred. As the triol, glycerol is preferred.

[0102] Examples of the fatty acid include formic acid, acetic acid, propionic acid, etc. Formic acid and acetic acid are preferred, and acetic acid is most preferred.

[0103] The ketone is not particularly limited, and preferably ketones having 3 to 6 carbon atoms can be used. Specific examples include acetone, methyl ethyl ketone, methyl butyl ketone, and methyl isobutyl ketone. Acetone and methyl ethyl ketone are preferred, and acetone is most preferred.

[0104] The nitrile may be cyclic, acyclic, saturated or unsaturated, and is not particularly limited, but saturated nitriles are usually preferably used. Nitriles having 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms, and more preferably 2 to 8 carbon atoms can be used.

[0105] Specific examples of the nitrile include acetonitrile, propionitrile, malononitrile, butyronitrile, isobutyronitrile, succinonitrile, valeronitrile, glutaronitrile, capronitrile, octanonitrile, nonononitrile, undecanonitrile, dodecanonitrile, tridecanonitrile, pentadeconitrile, stearonitrile, chloroacetonitrile, bromoacetonitrile, chloropropionitrile, bromopropionitrile, methoxyacetonitrile, methyl cyanoacetate, ethyl cyanoacetate, methylbenzonitrile, benzonitrile, chlorobenzonitrile, bromobenzonitrile, cyanobenzoic acid, nitrobenzonitrile, anisenitrile, naphthonitrile, bromomethylbenzonitrile, methyl cyanobenzoate, methoxybenzonitrile, acetylbenzonitrile, naphthonitrile, cyanobiphenyl, phenylpropionitrile, phenylbutyronitrile, methylphenylacetonitrile, diphenylacetonitrile, naphthylacetonitrile, nitrophenylacetonitrile, chlorophenylacetonitrile, cyclopropanecarbonitrile, cyclohexanecarbonitrile, cycloheptanecarbonitrile, phenylcyclohexanecarbonitrile, and tolylcyclohexanecarbonitrile.

[0106] Preferred are acetonitrile, propionitrile, succinonitrile, butyronitrile, isobutyronitrile, valeronitrile, methyl cyanoacetate, ethyl cyanoacetate, benzonitrile, methylbenzonitrile, and chloropropionitrile; more preferred are acetonitrile, propionitrile, butyronitrile, and isobutyronitrile; and most preferred is acetonitrile.

[0107] Examples of the nitrogen compounds other than nitriles include amides such as formamide, N-methylformamide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; nitromethane, triethylamine, and pyridine.

[0108] Examples of the sulfur compounds include dimethyl sulfoxide and sulfolane.

[0109] The above-mentioned organic solvent used in the present invention is preferably selected in consideration of properties such as boiling point, melting point, and viscosity. For example, as a boiling point, from the viewpoint that it can be appropriately heated to improve solubility and that solvent recovery and replacement can be easily performed, an organic solvent in the range of about 30 to 150° C. at 1 atmosphere is preferably used. As a melting point, from the viewpoint that it is not easy to solidify when operated at room temperature and cooled to below room temperature, an organic solvent of about 0° C. or more, preferably about 10° C. or more, and more preferably about 20° C. or more can be used. It is preferred to use an organic solvent with a viscosity as low as about 10 cp or less at 20° C.

[0110] In the production method of the present invention, when extracting coenzyme Q10 from an aqueous concentrated suspension of microbial cells or microbial cell disruptors, it is preferred to use a hydrophobic organic solvent or an organic solvent containing a hydrophobic organic solvent as the extraction solvent among the above-mentioned organic solvents. In addition, by using an organic solvent in which a small amount of a hydrophilic organic solvent (for example, alcohols such as isopropyl alcohol) and a surfactant are mixed in a hydrophobic organic solvent, the extraction efficiency can be further improved.

[0111] The hydrophobic organic solvent used in this case is not particularly limited. Among the above-mentioned organic solvents, hydrophobic solvents can be used, and hydrocarbons, fatty acid esters, and ethers are preferred, fatty acid esters or hydrocarbons are more preferred, and aliphatic hydrocarbons are further preferably used.

[0112] Among the above-mentioned aliphatic hydrocarbons, aliphatic hydrocarbons having 5 to 8 carbon atoms are preferably used. Specific examples of the above-mentioned aliphatic hydrocarbons having 5 to 8 carbon atoms include pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, 2-methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane, octane, 2,2,3-trimethylpentane, isooctane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, etc. Hexane, heptane, and methylcyclohexane are particularly preferred, and hexane is most preferred.

[0113] In addition, ethyl acetate can be preferably used as the fatty acid ester.

[0114] In the production method of the present invention, the amount of the extraction solvent is not particularly limited, and the concentration during extraction is preferably used in the range of 25 to 80 volume %, more preferably in the range of 50 to 75 volume %, relative to the volume of the total solution. In the production method of the present invention, the temperature during extraction is not particularly limited, and can usually be implemented in the range of 0 to 60° C., preferably 20 to 50° C.

[0115] As the above-mentioned extraction method, any method in batch extraction and continuous extraction can be used to carry out. Industrially, from the perspective of productivity, continuous extraction is preferred. In continuous extraction, countercurrent multi-stage extraction is particularly preferred. The stirring time during batch extraction is not particularly limited, and is usually more than 5 minutes. The average residence time during continuous extraction is not particularly limited, and is usually more than 10 minutes.

[0116] (5) Separation and removal of solid components

[0117] In the production method of the present invention, the extract of the coenzyme Q10-producing microorganism obtained as described above is directly cooled to precipitate the solid components, and the solid components are separated and removed; or, it is contacted with an alkaline aqueous solution to saponify the fat-soluble components from the microorganisms, and then concentrated after washing with water to prepare a concentrated extract, and then cooled to precipitate the solid components, and the solid components are separated and removed; thereby, impurities in the extract are removed and an extract with high coenzyme Q10 purity can be obtained.

[0118] Examples of the alkaline aqueous solution used for saponifying the extract of the coenzyme Q10-producing microorganism include aqueous ammonia, aqueous sodium hydroxide, aqueous potassium hydroxide, aqueous lithium hydroxide, aqueous sodium carbonate, aqueous sodium hydrogen carbonate, aqueous magnesium oxide, aqueous calcium hydroxide, and aqueous sodium acetate. In view of the saponification efficiency, a strong base is preferred, and in view of economic efficiency, an aqueous sodium hydroxide solution and an aqueous potassium hydroxide solution are more preferred.

[0119] The amount of the alkaline aqueous solution in contact with the extract is not particularly limited, but is 1% by volume to 200% by volume, preferably 1% by volume to 30% by volume, and more preferably 1% by volume to 10% by volume, relative to the total volume of the extract.

[0120] As the contact method with the above-mentioned alkaline aqueous solution, it can be carried out by any method in batch mode or continuous mode. Industrially, from the productivity aspect, it is preferably continuous mode. In the continuous mode, considering the cleaning property, it is particularly preferably the co-current mode. The stirring time during the batch mode is not particularly limited, and is usually more than 1 minute. The average residence time during the continuous extraction is not particularly limited, and is usually more than 10 seconds.

[0121] The extract after contact with the alkaline aqueous solution is prone to decomposition of coenzyme Q10 due to heat, etc., and quality reduction due to dimer formation, so it is preferably washed with water. The amount of water contacted with the extract is not particularly limited, but is 1% by volume or more and 200% by volume or less, preferably 1% by volume or more and 30% by volume or less, and more preferably 1% by volume or more and 10% by volume or less, relative to the total extract.

[0122] As the above-mentioned contact method, any method in a batch mode or a continuous mode can be used. In industry, from the perspective of productivity, a continuous mode is preferred. In the continuous mode, a parallel flow mode is particularly preferred in view of the cleaning property. The stirring time during the batch mode is not particularly limited, and is usually more than 1 minute. The average residence time during continuous extraction is not particularly limited, and is usually more than 10 seconds.

[0123] In the production method of the present invention, the microbial cell suspension containing the coenzyme Q10-producing microorganism concentrated by the porous membrane is concentrated through the above operation, and then prepared into a microbial cell fragment or an aqueous suspension of the microbial cell fragment, dried microbial cells or dried microbial cell fragment as needed, and then coenzyme Q10 is extracted in an organic solvent, and further contacted with an alkaline aqueous solution or water as needed, thereby separating / recovering the purified or purified coenzyme Q10.

[0124] The coenzyme Q10 solution after the water washing treatment can be used directly, or an adsorbent can be used to further remove impurities. The purified coenzyme Q10 extract can be further treated to prepare a composition containing high-purity coenzyme Q10 and coenzyme Q10 crystals as a more preferred method. As such a treatment process, it can be listed as: concentration, solvent replacement, oxidation, reduction, column chromatography, crystallization, etc., and of course they can also be combined. For example, the solvent can be distilled off (concentrated) from the coenzyme Q10 extract obtained by separation of the adsorbent to prepare a purified product containing coenzyme Q10, or after further purification by column chromatography such as silica gel as needed, the organic solvent is distilled off to prepare a purified product containing coenzyme Q10. In addition, the target coenzyme Q10 can also be obtained in the form of crystals by crystallization operations, etc.

[0125] Before the above-mentioned column chromatography, oxidation, reduction and crystallization, solvent replacement may be further performed as needed.

[0126] It should be noted that in the production method of the present invention, in order to produce reduced coenzyme Q10 alone or coenzyme Q10 with a high reduced coenzyme Q10 ratio as coenzyme Q10, it is effective to use a microorganism with a high reduced coenzyme Q10 content ratio in the produced coenzyme Q10 as a coenzyme Q10-producing microorganism, and perform extraction and purification treatment in an oxidation-resistant gas atmosphere (for example, an inert gas atmosphere such as nitrogen) after the above-mentioned concentration step. In this way, it is possible to obtain reduced coenzyme Q10 alone or coenzyme Q10 with a high reduced coenzyme Q10 ratio without performing special treatment. Of course, by further reducing the coenzyme Q10 with a high reduced coenzyme Q10 ratio obtained in this way, the reduced ratio can be further increased. Alternatively, the extract containing coenzyme Q10 may be produced without any particular method for preventing oxidation, or after being oxidized by oxygen in the air or an oxidant to obtain an extract having a low reduced coenzyme Q10 ratio (e.g., 50 mol % or less, or 30 mol % or less), by subjecting it to a reduction reaction, thereby producing coenzyme Q10 with a high reduced coenzyme Q10 ratio.

[0127] In order to produce reduced coenzyme Q10, it is preferred that the reduced coenzyme Q10 content ratio in the final step of production or as the final product is high. For example, the reduced coenzyme Q10 content in 100 mol% of the total amount of coenzyme Q10 can be 70 mol% or more, preferably 80 mol% or more, more preferably 90 mol% or more, and even more preferably 96 mol% or more.

[0128] As a more specific method, a culture suspension of a coenzyme Q10-producing microorganism can be passed through a porous membrane to be concentrated, and coenzyme Q10 can be extracted from the concentrate or its crushed product into an organic solvent to obtain an extract containing coenzyme Q10. After further purification using column chromatography, reduction treatment is performed, and crystallization operation is used to obtain high-purity reduced coenzyme Q10 crystals.

[0129] In addition, the production method of the present invention can also be used for the production of oxidized coenzyme Q10. In this case, coenzyme Q10 is extracted from a microbial cell concentrated suspension obtained by concentrating a coenzyme Q10-producing microbial suspension, a microbial cell fragment, or an aqueous suspension of a microbial cell fragment, dried microbial cells, or a dried microbial cell fragment into an organic solvent, and then an oxidation treatment using an oxidant can be performed; or, by performing extraction, adsorption, other purification, post-treatment, etc., only in the air, or by drying the bacterial cells in the air before extraction, coenzyme Q10 with a high ratio of oxidized coenzyme Q10 can be obtained by natural oxidation with a simple operation.

[0130] As a specific method, a culture suspension of a coenzyme Q10-producing microorganism can be passed through a porous membrane for concentration, and coenzyme Q10 can be extracted from the concentrate or its crushed product into an organic solvent. The obtained extract containing coenzyme Q10 can be subjected to solvent replacement and further purified using column chromatography, and then subjected to oxidation treatment and crystallization operation to obtain high-purity crystals of oxidized coenzyme Q10.

[0131] This application claims the benefit of priority of Japanese Patent Application No. 2018-087146 filed on April 27, 2018. The entire contents of the specification of Japanese Patent Application No. 2018-087146 filed on April 27, 2018 are incorporated herein by reference.

[0132] Example

[0133] The present invention is further described in detail by way of examples and comparative examples below, but the present invention is not limited to these examples. In addition, the yield of coenzyme Q10 and the purity of coenzyme Q10 in the examples and comparative examples are not limited to the limiting values ​​of the present invention, nor are the upper limits thereof.

[0134] The concentration of coenzyme Q10 was measured using high performance liquid chromatography (HPLC) (manufactured by SHIMADZU) under the following conditions.

[0135] (HPLC measurement conditions)

[0136] Column: YMC-Pack ODS-A

[0137] Oven temperature: 30℃

[0138] Mobile phase: methanol / hexane = 85 / 15 (volume ratio)

[0139] Liquid delivery speed: 1.0ml / min

[0140] Detection: UV275nm

[0141] The concentration when filtered through a porous membrane was calculated by directly calculating the amount of filtrate or measuring the coenzyme Q10 concentration of the coenzyme Q10-producing microbial suspension under the above-mentioned HPLC analysis conditions. In addition, the loss was evaluated by measuring the coenzyme Q10 concentration in the filtrate.

[0142] In addition, regarding the cleaning recovery of the porous membrane, water was passed through the porous membrane before passing the above-mentioned coenzyme Q10-producing microbial suspension, and the permeation rate after 3 hours was used as a benchmark. After filtration, warm water and reagents were used for cleaning, and then water was passed through the membrane in the same manner. The recovery rate was calculated based on the permeation rate after 3 hours.

[0143] Example 1

[0144] Saitoella complicata IFO10748 strain producing coenzyme Q10 was cultured aerobically at 25°C for 160 hours using a medium (5 g / L peptone, 3 g / L yeast extract, 3 g / L malt extract, 20 g / L glucose, pH 6.0).

[0145] Then, the obtained microbial culture solution containing coenzyme Q10 was heated to 60°C and adjusted to pH 6, and then the solution was passed through a porous membrane (φ = 6 mm, L = 609 mm, average pore diameter = 0.5 μm, filtration area = 0.0114 m) made of a hollow microporous body of titanium oxide and a stainless steel support at a linear speed of 4 m / s and a transmembrane pressure difference (TMP) of 0.2 MPa. 2 ; manufactured by Graver Co.) was passed through and filtered.

[0146] As a result of 10 hours of continuous operation, the solid content concentration before filtration (equivalent to the microbial concentration in the microbial cell suspension converted to the dry weight of microorganisms) was 8.06%, while it was concentrated to 10.35% after filtration. The average permeation flux through the filtration process was 0.69 kg / min / m 2 .

[0147] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0148] In addition, the microorganisms were crushed by a pressure crusher, and hexane in an amount equivalent to 1.8 times the volume of the microorganism crushed liquid and 2-propanol in an amount equivalent to 0.7 times the volume of the microorganism crushed liquid were added to the microorganism crushed liquid, and the operation of stirring at 40°C for 1 hour was repeated twice, and coenzyme Q10 was extracted by a two-step batch extraction operation. As a result, the extraction rate was 96.8%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0149] Example 2

[0150] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 6, and then filtered through a porous membrane at a linear velocity of 3 m / s and a transmembrane pressure difference (TMP) of 0.3 MPa in the same manner as in Example 1.

[0151] As a result of 10 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 10.32%. The average permeation flux through the filtration process was 0.59 kg / min / m 2 .

[0152] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0153] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 97.1%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0154] Example 3

[0155] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 40°C and adjusted to pH 6, and then filtered through a porous membrane at a linear velocity of 5 m / s and a transmembrane pressure difference (TMP) of 0.4 MPa in the same manner as in Example 1.

[0156] After 11.2 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 10.85%. The average permeation flux through the filtration process was 0.78 kg / min / m 2 .

[0157] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0158] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 97.0%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0159] Example 4

[0160] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 60°C, adjusted to pH 5, and then filtered through a porous membrane at a linear velocity of 3 m / s and a transmembrane pressure difference (TMP) of 0.4 MPa in the same manner as in Example 1.

[0161] After 10 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 10.27%. The average permeation flux through the filtration process was 0.55 kg / min / m 2 .

[0162] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0163] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 94.6%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0164] Example 5

[0165] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 4, and then filtered through a porous membrane at a linear velocity of 4 m / s and a transmembrane pressure difference (TMP) of 0.4 MPa in the same manner as in Example 1.

[0166] After 8.5 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 11.0%. The average permeation flux through the filtration process was 1.09 kg / min / m 2 .

[0167] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0168] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 97.2%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0169] Example 6

[0170] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 60°C, adjusted to pH 4, and then filtered through a porous membrane at a linear velocity of 5 m / s and a transmembrane pressure difference (TMP) of 0.3 MPa in the same manner as in Example 1.

[0171] After 6 hours of continuous operation, the solid content concentration before filtration was 8.15%, and after filtration it was concentrated to 13.04%. The average permeation flux through the filtration process was 1.60 kg / min / m 2 .

[0172] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0173] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 96.9%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0174] Example 7

[0175] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 40°C, adjusted to pH 4, and then filtered through a porous membrane at a linear velocity of 3 m / s and a transmembrane pressure difference (TMP) of 0.2 MPa in the same manner as in Example 1.

[0176] After 10 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 10.09%. The average permeation flux through the filtration process was 0.65 kg / min / m 2 .

[0177] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0178] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 96.9%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0179] Example 8

[0180] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then filtered through a porous membrane at a linear velocity of 5 m / s and a transmembrane pressure difference (TMP) of 0.2 MPa in the same manner as in Example 1.

[0181] After 8.7 hours of continuous operation, the solid content concentration before filtration was 8.06%, and after filtration it was concentrated to 11.01%. The average permeation flux through the filtration process was 1.08 kg / min / m 2 .

[0182] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0183] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 95.9%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0184] Example 9

[0185] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 4, and then filtered through a porous membrane at a linear velocity of 5 m / s and a transmembrane pressure difference (TMP) of 0.2 MPa in the same manner as in Example 1.

[0186] After 6.7 hours of continuous operation, the solid content concentration before filtration was 7.9%, and after filtration it was concentrated to 12.2%. The average permeation flux through the filtration process was 1.65 kg / min / m 2 .

[0187] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0188] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 98.0%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0189] Example 10

[0190] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 4, and then filtered through a porous membrane (φ = 9 mm, L = 1520 mm, average pore diameter: 0.5 μm, filtration area: 0.0462 m) made of a hollow microporous body of titanium oxide and a stainless steel support at a linear velocity of 5 m / s and a transmembrane pressure difference (TMP) of 0.2 MPa. 2 ; manufactured by Graver Co.) was passed through and filtered.

[0191] After 29.5 hours of continuous operation, the solid content concentration before filtration was 7.11%, and after filtration was 12.96%. The average permeation flux through the filtration process was 2.35 kg / min / m 2 .

[0192] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0193] The obtained concentrated microorganism suspension was crushed by pressure to extract coenzyme Q10 in the same manner as in Example 1. As a result, the extraction rate was 98.0%, and it was confirmed that coenzyme Q10 was well extracted by concentration using a porous membrane.

[0194] Embodiment 11

[0195] In the filtration operation of Example 10, air was flowed in from the filtrate discharge side for 30 seconds to clean and regenerate the porous membrane. As a result, the permeation flux before regeneration was 1.49 kg / min / m 2 In contrast, after regeneration, it recovered to 2.34kg / min / m 2 .

[0196] Example 12

[0197] After the filtration operation of Example 11, the porous membrane was circulated and washed with warm water at 50°C for 1 hour, and the washing recovery was 40%. In addition, the concentration of coenzyme Q10 in the washing solution used for washing was measured and found to be below the detection limit.

[0198] Embodiment 13

[0199] After the above Example 12, the porous membrane was circulated and cleaned with a 2% sodium hydroxide aqueous solution at 70° C. for 1 hour. As a result, the cleaning recovery was 97%.

[0200] In addition, the concentration of coenzyme Q10 in the cleaning solution used for cleaning was measured and the result was below the detection limit.

[0201] Embodiment 14

[0202] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then filtered through a porous membrane at a linear velocity of 10 m / s and a transmembrane pressure difference (TMP) of 0.25 MPa in the same manner as in Example 1.

[0203] After 7.5 hours of continuous operation, the solid content concentration before filtration was 6.96%, and after filtration it was concentrated to 13.29%. The average permeation flux through the filtration process was 3.41 kg / min / m 2 .

[0204] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0205] Embodiment 15

[0206] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then passed through a porous membrane in the same manner as in Example 1. The linear velocity was changed to 7 m / s for 4 hours, then 5 m / s for 5.5 hours, and then 6 m / s for 4 hours. The membrane pressure difference (TMP) was set to 0.35 MPa, and filtration was performed.

[0207] The continuous operation was performed for a total of 13.5 hours. The result was that the solid content concentration before filtration was 7.46%, while that after filtration was 12.71%. The average permeation flux through the filtration process was 2.74 kg / min / m 2 .

[0208] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0209] Example 16

[0210] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then filtered through the porous membrane at a linear velocity of 7 m / s and a transmembrane pressure difference (TMP) of 0.25 MPa in the same manner as in Example 1. The solid content concentration before the filtration treatment was 6.71%. When it was confirmed that it was concentrated to 12.5%, a portion of the obtained filtrate was returned to the original solution of the microbial culture suspension before the treatment, the solid content concentration was diluted to 9.5%, and concentrated again to 12.5%. Such continuous repeated tests were performed a total of 4 times.

[0211] The average permeation flux of the first concentration was 2.41 kg / min / m 2 , 2nd time 2.40kg / min / m2 3rd time: 2.08kg / min / m 2 4th time 1.38kg / min / m 2 Although the average permeation flux gradually decreased, the filtration was well performed through four repeated runs.

[0212] Embodiment 17

[0213] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then filtrated onto a ceramic membrane (φ = 3.5 mm, L = 1187 mm, average pore diameter: 0.2 μm, filtration area: 0.35 m) at a linear velocity of 7.5 m / s and a transmembrane pressure difference (TMP) of 0.19 MPa. 2 ; manufactured by TAMI Co., Ltd.) was passed through and filtered.

[0214] After 1.5 hours of continuous operation, the solid content concentration before filtration was 8.0%, while after filtration it was 12.84%. The average permeation flux through the filtration process was 2.92 kg / min / m 2 .

[0215] In addition, the coenzyme Q10 concentration on the filtrate side was measured and found to be below the detection limit.

[0216] Embodiment 18

[0217] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 50°C, adjusted to pH 5, and then filtered through the ceramic membrane at a linear velocity of 7 m / s and a transmembrane pressure difference (TMP) of 0.3 MPa in the same manner as in Example 17.

[0218] After 30 minutes of continuous operation, the solid content concentration before filtration was 7.57%, while after filtration it was 12.93%. The average permeation flux through the filtration process was 3.81 kg / min / m 2 Then, the cycle was switched and run for 20 hours. As a result, the average permeation flux during this period decreased to 1.32 kg / min / m 2 The average permeation flux observed during the entire run was 1.80 kg / min / m 2 .

[0219] (Comparative Example 1)

[0220] An attempt was made to filter the microbial culture solution (solid content concentration 8.06%) containing coenzyme Q10 obtained in the same manner as in Example 1 using a Kiriyama funnel and filter paper No. 5-C for a Kiriyama funnel, but clogging occurred from the beginning and no filtrate could be obtained.

[0221] (Comparative Example 2)

[0222] The microbial culture solution containing coenzyme Q10 (solid content concentration 8.06%) obtained in the same manner as in Example 1 was centrifuged at 1000 g for 5 minutes using Allegra X-22R CENTRIGUGE manufactured by BECKMAN COULTER to separate into a microbial concentrate and a supernatant, and the supernatant was recovered. It was confirmed that the coenzyme Q10 concentration in the supernatant was 0.3 g / L, and 1.4% of the coenzyme Q10 in the microbial culture solution was lost.

[0223] (Comparative Example 3)

[0224] The microbial culture solution containing coenzyme Q10 (solid content concentration 8.06%) obtained in the same manner as in Example 1 was centrifuged at 2000 g for 5 minutes using the same centrifuge as in Comparative Example 2 to separate into a microbial concentrate and a supernatant, and the supernatant was recovered. It was confirmed that the coenzyme Q10 concentration in the supernatant was 0.1 g / L, and 0.6% of the coenzyme Q10 in the microbial culture solution was lost.

[0225] (Comparative Example 4)

[0226] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 30°C, adjusted to pH 5, and then filtrated onto a ceramic membrane (φ = 6 mm, L = 1187 mm, average pore diameter: 0.2 μm, filtration area: 0.16 m) at a linear velocity of 3 m / s and a transmembrane pressure difference (TMP) of 0.05 MPa. 2 ; manufactured by TAMI Co., Ltd.) was passed through and filtered.

[0227] After 4.5 hours of continuous operation, the solid content concentration before filtration was 7.0%, while after filtration it was concentrated to 11.72%, and the average permeation flux through the filtration process was significantly reduced to 0.49 kg / min / m 2 .

[0228] (Comparative Example 5)

[0229] The microbial culture solution containing coenzyme Q10 obtained in the same manner as in Example 1 was heated to 30°C, adjusted to pH 5, and then filtrated onto an organic membrane (average pore diameter: 0.2 μm, filtration area: 0.022 m) at a linear velocity of 1.1 m / s and a transmembrane pressure difference (TMP) of 0.015 MPa.2 ; manufactured by DAISEN Co., Ltd.) was passed through and filtered.

[0230] After 12 hours of continuous operation, the solid content concentration before filtration was 6.75%, while it was concentrated to 9.98% after filtration. The average permeation flux through the filtration process was significantly reduced to 0.42 kg / min / m 2 .

Claims

1. A method for producing coenzyme Q10, the method comprising: A filtration step of passing a culture suspension of a coenzyme Q10-producing microorganism through a porous membrane while being heated to a temperature of 35° C. to 90° C. to obtain a microbial cell culture concentrate having a higher solid content concentration than that of the culture suspension; an extraction step of disrupting the microbial cells in the microbial cell culture concentrate to obtain a microbial cell disrupted product or an aqueous suspension of the microbial cell disrupted product, and extracting coenzyme Q10 from the disrupted product or the aqueous suspension of the microbial cell disrupted product; The porous membrane is any one of a synthetic resin, ceramic, or metal membrane. The average micropore diameter of the porous membrane is 0.01 to 3 μm. The concentration of coenzyme Q10 in the filtrate obtained through the filtration step was below the detection limit.

2. The manufacturing method according to claim 1, wherein: The heating temperature is above 48°C.

3. The manufacturing method according to claim 1 or 2, wherein: The pH of the culture suspension is in the range of 3-7.

4. The manufacturing method according to claim 1 or 2, wherein: The linear velocity of the liquid in the filtering step is above 0.1 m / s.

5. The manufacturing method according to claim 1 or 2, wherein: In the liquid passing treatment of the culture suspension, a regeneration treatment of sealing the filtration device, pressurizing the filtration device, and then releasing the pressure is performed at least once.

6. The manufacturing method according to claim 5, wherein: The pressure during the pressurization is 0.1 to 1 MPa.

7. The manufacturing method according to claim 5, wherein: The medium used for the pressurization is any one or more of air, nitrogen, water, the culture suspension, and a filtrate obtained by passing the culture suspension through a porous membrane.

8. The manufacturing method according to claim 1 or 2, wherein: After the filtration step is completed, the following operations are repeated: a washing step of washing the microbial suspension components attached to the porous membrane, and then the filtration step is performed again.

9. The manufacturing method according to claim 8, wherein: The cleaning liquid used for the cleaning is any one or more of water, an alkaline aqueous solution, and an acidic aqueous solution.

10. The manufacturing method according to claim 8, wherein: The temperature of the cleaning liquid for the cleaning is 10°C to 90°C.

11. The manufacturing method according to claim 1, wherein: The porous membrane is a metal porous membrane.

12. The manufacturing method according to claim 11, wherein: The metal porous membrane is a tube including a separation layer based on titanium oxide and a stainless steel support, and has an inner diameter of 5 mm or more.

13. The manufacturing method according to claim 1 or 2, wherein: In the liquid passing treatment of the culture suspension, after the liquid passing treatment is performed by flowing the filtrate into the medium from the outlet side of the filtrate, the filtration step is resumed.

14. The manufacturing method according to claim 13, wherein: The medium to be flowed in is any one or more of air, nitrogen, water, and a filtrate obtained by passing the culture suspension through a porous membrane.

15. The manufacturing method according to claim 13, wherein: The temperature of the inflowing medium is 10°C to 90°C.